1. Field of the Invention
The invention is related to the detection of West Nile Virus (WNV) infections in animals.
2. Description of Related Art
Known as a Flavivirus, the West Nile virus was first identified in 1937 in Africa and first found in North America in 1999. Migratory birds are considered the primary means whereby infection is spread within and between countries. The virus is transmitted by mosquitoes that have acquired infection by feeding on viremic birds. The virus is then amplified during periods of adult mosquito blood-feeding. Infected mosquitoes then transmit the virus to humans and animals upon feeding thereon.
West Nile virus is the causative agent for West Nile Virus disease, particularly West Nile encephalitis, predominately in humans, other mammals and birds. The chief concern in both the United States and foreign countries is the lack of effective treatment for West Nile virus disease. Anti-inflammatory drugs are used to combat swelling of central nervous system tissues, but beyond that no medical intervention is currently available.
The West Nile fever virus also affects horses, particularly in North America and Europe (Cantile C. et al., Equine Vet. J., 2000, 32 (1), 31–35). These horses reveal signs of ataxia, weakness of the rear limbs, paresis evolving towards tetraplegia and death. Horses and camels are the main animals manifesting clinical signs in the form of encephalitis.
The virions of the West Nile fever virus are spherical particles with a diameter of 50 nm constituted by a lipoproteic envelope surrounding an icosahedric nucleocapsid containing a positive polarity, single-strand RNA. A single open reading frame (ORF) encodes all the viral proteins in the form of a polyprotein. The cleaving and maturation of this polyprotein leads to the production of several different viral proteins. The structural proteins are encoded by the 5′ part of the genome and correspond to the nucleocapsid designated C (14 kDa), the envelope glycoprotein designated E (50 kDa), the pre-membrane protein designated prM (23 kDa), and the membrane protein designated M (7 kDa). The non-structural proteins are encoded by the 3′ part of the genome and correspond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa), NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), and NS5 (97 kDa).
Vaccines for WNV are described, for example, in U.S. Patent Publication Nos. 2003/0148261A1, 2003/0104008A1 and 2003/0091595A1, each of which is incorporated herein by reference in its entirety. Publication No. 2003/0091595 describes a WNV vaccine that includes an inactivated whole or subunit WNV. Publication No. 2003/0104008 discloses a vector, such as recombinant avipox virus, containing and expressing exogenous polynucleotide(s) from WNV to induce an immune response against WNV. These recombinant WNV vaccines include a vector containing a polynucleotide having single encoding frame corresponding to, for example, prM-E, M-E and prM-M-E. The vector may include several separate polynucleotides encoding the different proteins (e.g. prM and/or M and E). The vector can also include polynucleotides corresponding to more than one WN virus strain, for example, two or more polynucleotides encoding E or prM-M-E of different strains. Furthermore, the vector can include one or more nucleotide sequences encoding immunogens of other pathogenic agents and/or cytokins. Publication No. 2003/0148261 describes various WNV polypeptides and immunogenic fragments for use in WNV vaccines. These vaccines are produced recombinantly using various vectors encoding WNV polypeptides and the vectors are expressed by a variety of host cells.
Various method for detecting WNV are known. E. g., Feinstein, S, et al., Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA), J Med Virol. 1985; 17(1):63–72; Martin, D A, et al., Standardization of Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assays for Routine Diagnosis of Arboviral Infections, J. Clin. Micro., 2000; 38(5): 1823–1826. Martin et al describe the currently accepted CDC method, known generally as a MAC-ELISA, for detecting IgM produced by an animal in response to an exposure to a WNV antigen. Generally, this method involves contacting goat anti-IgM coated plates with a sample and incubating the plate overnight. Following the incubation, the plate, having bound IgM from the sample, is then contacted with WNV antigen from virus infected suckling mouse brain or unpurified C6/36 cell culture supernates. This is followed by the addition of flavivirus group reactive monoclonal antibody conjugated to HRP and a further incubation. Bound conjugate is detecting by adding TMB substrate and A450 is measured.
The CDC MAC-ELISA takes between 24 and 72 hours to complete, with at least one overnight incubation. While this assay can effectively detect exposure to a WNV antigen, the assay cannot detect whether a positive result is due to a natural WNV infection or vaccination of the animal. Therefore, asymptomatic animals that have an unknown vaccination status testing positive for the MAC-ELISA may incorrectly be diagnosed as positive for WNV infection when, in fact, the animal has developed antibodies to WNV as a result of vaccination. Thus, what is needed is a rapid method of detecting exposure to a WNV antigen. Also, a method is needed that can distinguish between animals that have been naturally infected with WNV from animals that have been vaccinated.